The outer membrane of mitochondria contains pore‐forming beta‐barrel proteins, which facilitate the diffusion of molecules of up to about 3,000 Dalton across the outer membrane. Beta‐barrel proteins are also found in the outer membranes of bacteria and chloroplasts, revealing the common evolutionary origin. Owing to this bacterial descent, mitochondrial beta‐barrel proteins are integrated from the ‘inside’, that is, from the intermembrane space (IMS). After being translocated through the TOM complex, precursors of beta‐barrel proteins are stitched into the outer membrane in an export‐like process by the sorting and assembly machinery (SAM) complex. Cryo‐electron microscopy structures of the SAM complex, in some cases with associated translocation intermediates, have recently revealed fascinating details of this machinery [5, 6, 7, 8]. Nils Wiedemann, Nikolaus Pfanner, and coworkers provide a comprehensive overview of the structure and function of the beta‐barrel assembly machinery [9].

During the evolution of eukaryotic cells, the IMS of mitochondria was derived from the periplasmic space of the endosymbiont. Presumably owing to the same evolutionary descent, soluble proteins of the bacterial periplasm and of the IMS reach their three‐dimensional structure by oxidative protein folding. The review by Christine Zarges and Jan Riemer describes the enzymes of the human IMS, which insert disulfide bonds into newly imported proteins [10]. The two major enzymes, the FAD‐bound sulfhydryl oxidase ALR/Erv1 and oxidoreductase CHCHD4/Mia40, are conserved from yeast to human. However, in humans, Mia40 is tethered to the inner membrane by the NADH dehydrogenase AIF adding an interesting regulatory component to the human mitochondrial disulfide relay.

Most mitochondrial proteins reside in the matrix. These proteins are synthesized in the cytosol with aminoterminal presequences to direct them into mitochondria. The presequence translocase of the inner membrane (also known as the TIM23 complex) facilitates their translocation across the inner membrane. Recent structures of the TIM23 complex of yeast showed that Tim17 represents the protein‐conducting essential subunit contradicting previous assumptions [11, 12]. Tim17 forms a half‐channel that is partially exposed to the lipids of the inner membrane and uses negatively charged residues to promote the passage of the positively charged presequences into the matrix [13]. In this ‘In the Limelight’ special issue of FEBS Open Bio, Agnieszka Chacinska and coworkers show models of the human TIM23 translocase and compare its structure to that of yeast cells [14].